POU4F1 Promotes the Primary Resistance of Melanoma to Anti‐PD‐1 Therapy by Regulating Glycolysis Through METTL1‐Mediated m7G Methylation of PKM2

ABSTRACT Melanoma, a highly malignant tumor originating from melanocytes, has seen a significant increase in global incidence, particularly among the elderly. Anti‐PD‐1 monoclonal antibodies, which activate the immune system to attack cancer cells by blocking the PD‐1/PD‐L1 signaling pathway, have improved survival rates but face challenges such as innate resistance. This study enrolls 37 melanoma patients and 7 benign nevus patients, with tissue samples collected for analysis. RT‐qPCR and Western blot are used to quantify the expression of the target protein. Flow cytometry is utilized to analyze immune subsets in tumors. Glucose uptake, lactate production, and ATP level are assessed by commercial kits. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) are measured using a Seahorse XF Analyzer. RNA Bisulfite Sequencing is performed to quantify pyruvate kinase M2 (PKM2) m7G methylation level, MeRIP‐qPCR is conducted to validate the m7G methylation level of PKM2, and RIP assay is conducted to confirm POU4F1‐METTL1 interaction. The results show that POU Class 4 Homeobox 1 (POU4F1) is upregulated in melanoma tissues compared to benign nevi. Anti‐PD‐1 treatment effectively reduces POU4F1 expression in sensitive B16‐F10‐M tumors and has no significant effect on resistant B16‐F10‐R tumors. POU4F1 overexpression induces profound metabolic alterations, including increased lactate production, glucose uptake, and ECAR, while suppressing OCR in B16‐F10‐M cells. POU4F1 overexpression also reduces the infiltration of CD8 + T cells, M1‐macrophages, and NK cells, while increasing Treg and M2‐macrophage populations in B16‐F10‐M cells. Importantly, 3‐Bromopyruvate (a glycolysis inhibitor) reverses these effects. Mechanistically, POU4F1 upregulates METTL1 expression and increases m7G methylation of PKM2 mRNA. Besides, there is an interaction between POU4F1 and METTL1. METTL1 is also overexpressed in melanoma tissues compared to benign nevi. In conclusion, POU4F1 drives anti‐PD‐1 resistance in melanoma by enhancing glycolysis via METTL1‐mediated m7G methylation of PKM2. Targeting the POU4F1‐METTL1‐PKM2 axis may improve melanoma immunotherapy outcomes.